Multi-CBV vaccine for preventing or treating type I diabetes

ABSTRACT

The invention is directed to a vaccine comprising: i) coxsackie B virus CBV1 and CBV2, and ii) at least one coxsackie B virus selected from CBV3, CBV4, CBV5 and CBV6. The CBVs are present in the vaccine in inactivated form, in the form of a component of the virus or as an antibody against the virus. The vaccine is effective in preventing and treating type 1 diabetes. So is an anti-coxsackie B virus composition provided.

CROSS REFERENCE

This application is a continuation of U.S. patent application Ser. No.15/465,854, filed Mar. 22, 2017, which is a continuation of U.S. patentapplication Ser. No. 14/352,585, filed Apr. 17, 2014, abandoned, whichis a national stage filing under 35 U.S.C. § 371 of PCT/EP2012,070653,which claims priority to EP 11306344.0, filed Oct. 18, 2011, whichapplications are incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The present invention relates to anti-viral compositions e.g. vaccinesand methods useful in preventing or treating type 1 diabetes. Moreparticularly the invention relates to a vaccine comprisingenteroviruses, components thereof and/or antibodies thereto.

BACKGROUND OF THE INVENTION

Type 1 diabetes (T1D) is a severe disease that has become more and morefrequent already at a very early age. In type 1 diabetes the beta-cellsof the pancreas are destroyed, which leads to insulin deficiency. Thedestruction of the beta-cells is believed to be caused by an autoimmuneresponse, which in turn is assumed to be induced by a virus infection.

The connection between enteroviruses and T1D has been documented in amultitude of studies. Enteroviruses have been detected in the pancreas,blood and intestinal mucosa of patients with T1D more frequently than incontrol subjects and prospective studies have supported their role inthe initiation of the beta-cell damaging process associated with T1D.Enteroviruses infect pancreatic beta-cells in cell culture and causediabetes in animal models. Enterovirus vaccines have therefore beensuggested for preventing T1D. However, the knowledge of whichenterovirus serotypes that are involved in the disease is limited.

The group of enteroviruses includes more than 100 different serotypes,and because a vaccine covering all the 100 enterovirus serotypes is notrealistic using the current standard vaccine technologies, the knowledgeof which serotypes are involved in T1D is critical for vaccinedevelopment. Enterovirus infections are usually subclinical, but theymay also cause various kinds of diseases. For example polioviruses,coxsackie B virus (CBV), coxsackie A virus (CAV), and echovirus as wellas numbered enteroviruses are enteroviruses known to be involved in thedevelopment of a variety of diseases. For example Lee et al., 2007, ArchVirol, 152:963-970 analyzed clinical isolates from aseptic meningitispatients, and found that CBV5 was the most predominant strain. Inaddition CBV1, CBV3 and echovirus 9 were identified.

The spectrum of responsible serotypes varies a lot from disease todisease, and even in one disease like T1D the exact serotypes have notbeen fully and reliably identified. Indeed, previous studies reportingassociation between enteroviruses and T1D were not able to discriminatebetween serotype or were restricted to case reports. Some studies havesuggested that a particular CBV serotype may be important (Yoon et al,1979 New Eng J of Med, v 300, p 1173; Hindersson M, et al., 2005, J ClinVirol v.33 p 158; Dotta F. et al., 2007, PNAS 104: 5115; US2010/0047273(Rappuoli et al.)). However, other than CBV enteroviruses have also beensporadically reported (Cabrera-Rode et al., 2003, Diabetologia; Williamset al., 2006, J Clin Micro v.44 p 441).

Klemola et al., 2008, Immunology of Diabetes V: Ann. N.Y. Acad. Sci1150:210-212 analyzed sewage samples for non-polio enteroviruses. Themost commonly detected serotypes were CBV1-5, Echo6, 7, 11, 25 and 30.They were tested for islet tropism. All serotypes were found to bedestructive. No evidence suggesting that only certain enterovirusserotypes should be considered potentially diabetogenic was found.

WO01/00236 (Hyöty et al.) suggests the use of oral poliovirus in avaccine against T1D. In addition it suggests another diabetes vaccinecomprising one or more inactivated non-polio enteroviruses selected fromthe group consisting of CBV serotypes 1, 2, 3, 4, 5 and 6, echovirusserotypes 3, 4, 6, 9, 11, 22 and 30, CAV serotypes 9 and 16. However, nodata is given as to the role of the individual serotypes listed. Theimmune response obtained by OPV, which is a live attenuated vaccine wasbelieved to induce a T-cell mediated cross-reactive immune response,whereas the inactivated vaccine was believed to be serotype specific.

Despite a lot of investigation, there is still a great need fordeveloping effective means and methods for preventing and treating type1 diabetes. Embodiments of the present invention meet these needs.

SUMMARY OF THE INVENTION

The present invention is based on the finding that the whole coxsackie Bvirus (CBV) group may induce type 1 diabetes (T1D). Preliminary testsshowed that CBV1 and CBV2 were clear risk serotypes, whereas CBV3, CBV4,CBV5 and CBV6 seemed to reduce the risk of the disease. The inventionresides in the surprising finding that an even more effective vaccineagainst T1D should include not only those CBVs that show a high risk forinducing the disease, but also those serotypes that appear neutral oreven protective against T1D.

In addition, an antibody-mediated cross-reaction was found between thedifferent CBV serotypes. This finding makes it feasible to useantibody-inducing vaccines i.e. vaccines comprising inactivated viruses,or components thereof. These vaccines are safer than attenuated livevaccines, but still very effective. Alternatively the vaccine mayalready contain antibodies against the CBVs.

The present invention is directed to a vaccine comprising: i) coxsackieB virus CBV1 and CBV2 in inactivated form or in the form of a componentof said virus, and ii) at least one coxsackie B virus selected from thegroup consisting of CBV3, CBV4, CBV5 and CBV6 in inactivated form or inthe form of a component of said virus.

The present invention is also directed to a composition e.g. a vaccinecomprising: i) antibodies against coxsackie B virus CBV1 and CBV2, andii) antibodies against at least one coxsackie B virus selected from thegroup consisting of CBV3, CBV4, CBV5 and CBV6.

The present invention is further directed to said vaccines for use inpreventing or treating type 1 diabetes.

Still further the invention is directed to an anti-coxsackie B viruscomposition for use in treating or preventing type 1 diabetes.

A method for preventing or treating type 1 diabetes in a subject in needthereof comprising administering to the subject a vaccine comprising: i)coxsackie B virus CBV1 and CBV2 in inactivated form or in the form of acomponent of said virus, and ii) at least one coxsackie B virus selectedfrom the group consisting of CBV3, CBV4, CBV5 and CBV6 in inactivatedform or in the form of a component of said virus is described.

A method for preventing or treating type 1 diabetes in a subject in needthereof comprising administering to the subject a vaccine comprising: i)antibodies against coxsackie B virus CBV1 and CBV2, and ii) antibodiesagainst at least one coxsackie B virus selected from the groupconsisting of CBV3, CBV4, CBV5 and CBV6 is also described.

Further a method for preventing or treating type 1 diabetes in a subjectin need thereof comprising administering to the subject ananti-coxsackie B virus (anti-CBV) composition is described.

Some specific embodiments of the invention are set forth in thedependent claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the OR and CI values, and the prevalence of antibodies forthe CBV serotypes.

FIG. 2 shows a summary of the tight and loose criteria CBV1 time windowanalyses.

FIG. 3 shows the high risk of beta-cell autoimmunity in children aged1.5 years, who experience CBV1 infection in the absence of maternalantibodies.

FIG. 4 shows a combined population attributable risk (PAR) for CBV1 andCBV2.

FIG. 5 shows viremia in vaccinated and unvaccinated mice on Day 2 andDay 7 post challenge with live CBV1 with dose of 10⁴CCID₅₀ administratedvia ip route.

FIG. 6 shows virus quantification in pancreas in vaccinated andunvaccinated mice on Day 7 post challenge with live CBV1 with dose of10⁴CCID₅₀ administrated via ip route.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the new finding that all six differentcoxsackie B group viruses (CBVs) can be potential risk viruses for type1 diabetes (T1D) and therefore all the CBVs may be included into avaccine. This kind of vaccine is optimal for the prevention of T1D. EachCBV serotype included in the vaccine will also give protection againstnot only the homotypic virus type but also cross-protection against theother CBV types.

There are six genetically and structurally closely related CBV serotypesthat are the only known enteroviruses to use the cellular receptor CARto enter the target cell. CAR is expressed on human insulin-producingbeta cells in the pancreas and all CBVs productively infect beta cellsin vitro. The six CBVs can induce inter-serotype cross-protection. Sincethe six serotypes are the only enteroviruses to use CAR, they may sharesome specific characteristics, either in terms of antigenicity or interms of tropism.

There are recognized risk viruses such as CBV1 and CBV2 that wereassociated with increased risk of T1D in virus antibody screening study(causative viruses). In addition, certain other CBV serotypes such asCBV3 and CBV6 were associated with decreased risk of T1D (protectiveeffect) in these analyses. In additional analyses, the presence of theCBV group viruses in serum was associated with increased risk of T1Dsuggesting that all CBV viruses can have diabetogenic potential. Thediabetogenic potential may be first determined by the ability to infectbeta cells. It may then be linked to any CBV serotypes and thepathogenicity may be modulated by genetic mutations in CBV strains andrecombinations which are known to occur between CBV serotypescirculating in a given population. Such serotype and strain-specificmodulation of pathogenicity has been observed for polioviruses. Allthree serotypes can cause poliomyelitis because they are the onlyenteroviruses to use PVR as their receptor, however PV2 was generallyless pathogenic than PV1, and furthermore, for each serotypes, somestrains (e.g. the vaccine strains Sabin) showed a very weakpathogenicity.

In one population few CBV serotypes may predominate as diabetogenicviruses such as CBV1 and CBV2 in the Finnish population in the presentstudy, while other CBV serotypes may have a much weaker diabetogeniceffect. The viruses with a weaker diabetogenic potential would thenappear protective due to their ability to induce immunologicalcross-protection against more diabetogenic CBV serotypes.

A virus may also be diabetogenic in one part of a population but eitherprotective or neutral in another part of the same population. Suchphenomenon can be based for example on the different genetical make-upof different subpopulations such as differences in HLA, MDA-5 or otherimmunologically important genotypes. One of the clearest genetics drivenpopulation dividing characteristic is the sex, and therefore differentrisk, neutral or protective characters may be observed for a serotypewhen its influence is studied separately in females and males. Theprotective and risk character may also depend on the time of infection.The later the time window is the more risk character the virus shows, Onthe other hand, the infections at a very early age, without protectionprovided by maternal antibodies, can be extremely risky.

There are certain scenarios, by which the neutral or even protectivevirus types can turn into risk viruses or certain risk viruses can bereplaced with other ones:

An example of such a process is provided by polioviruses (belonging tothe enteroviruses). In this case the live attenuated vaccine strainsinduce an immunological response against its wild-type predecessor. Insome rare cases the attenuated vaccine strains can revert back to thedisease causing form and therefore it represents a mild risk for thedisease. Such strain can therefore be called “vaccine-derivedpoliovirus” (VDPV). Inter-serotype recombination is a mechanism that maylead to emergence of new risk viruses. This can occur betweengenetically related serotypes and it is a relatively common phenomenonespecially among closely related enteroviruses (Vopr Virusol. 2005May-June; 50(3):46-52). A special character of the CBVs is that they alluse the CAR receptor and infect pancreatic beta-cells in vitro. Theserological properties and receptor binding characteristics of CBVs aredetermined by their structural capsid proteins, the receptor bindingcharacteristic being crucial for the cellular tropism. However, theinfection dynamics or virulence is mainly determined by other regions ofthe CBV genome; for the latter ones the special importance are 5′ and3′-non-coding regions (NCRs) as well as non-structural protein encodingregions. Recombinations between enteroviruses occur usually betweenso-called hot-spots, which lie in the enterovirus genome between the5′-NCR and structural proteins and between structural proteins and anon-structural protein. Therefore, inter-serotype recombination can leadto mixing of the serological and receptor binding properties of onestrain with “pathological” properties of another enterovirus strain.Thus a multi-CBV vaccine will have a crucial role in preventing thistype of development among enteroviruses associated with T1D. “Multi-CBVvaccine” refers to a vaccine directed to more than one CBV serotype.

In HPV studies it has been found that when the strongest risk strainsare eliminated from the human populations by vaccination the releasedniche can be occupied by other disease causing strains (Int J Cancer.2011 Mar. 1; 128(5):1114-9. doi: 10.1002/ijc.25675. Epub 2010 Nov. 28.).A similar phenomenon can occur if the strongest CBV risk serotypes areeliminated by vaccination. In such populations where the strong riskserotypes are rare this can also occur naturally.

In view of the above, it is reasonable to include many or all of the sixCBV serotypes into the same vaccine in order to guarantee its maximalefficiency in terms of immune protection against diabetogenicenteroviruses.

The vaccine of the invention comprises the CBVs in inactivated form,and/or it comprises components of the viruses, and/or antibodies againstsaid viruses. In addition to the inactivated CBV1 and CBV2, componentsthereof, or antibodies thereto, the vaccine contains at least one, two,three or four of CBV3, CBV4, CBV5 and CBV6, components thereof, orantibodies thereto in any combination. Due to the antibody-mediatedcross-reactivity found between the CBVs, a protective effect against amissing serotype may also be obtained. However, in a preferredembodiment the vaccine is a “panCBV vaccine”, which means that it isdirected against all six serotypes, i.e. it comprises inactivated CBV1,CBV2, CBV3, CBV4, CBV5, and CBV6, and/or components of said six CBVs, orantibodies against said six CBVs. The antibody-mediated cross-reactivityfound between the CBVs is believed to induce a synergistic effect insuch a pan-CBV vaccine.

The forms of the CBVs present in the vaccine are independent from eachother. For example some of the CBVs may be present in inactivated formand others as components e.g. as virus like particles (VLPs) or assubunits. Conveniently all the CBVs present in the vaccine are in thesame form, e.g. inactivated, subunits, VLPs, nucleic acids orantibodies.

A “vaccine” is considered to be a composition capable of providing aprotective immune response in the recipient. The vaccine may eitherelicit an active immune response i.e. it induces a protective immuneresponse in the recipient, or it may provide a passive immune response,i.e. it already contains antibodies needed to prevent or treat thedisease.

The vaccine is administered in a “pharmaceutically effective amount”meaning an amount, which is able to elicit an immune response thatprotects the vaccine recipient against the enterovirus either byeliciting neutralizing antibodies or a cell-mediated response, or both.In case of antibodies a pharmaceutical effective amount is one thatmediates a protective immune response against the virus.

The vaccine may contain the virus in “inactivated” form, which meansthat the infectivity of the virus has been destroyed. The term as usedherein also includes viruses that are replication defective. Areplication defective virus is a virus defective for a constitutiveprotein. Such a virus can enter the cell, deliver the partial genome,translate the proteins encoded and replicate the genome but cannotencapsidate new particles. Thus it cannot spread to neighboring cells ortissue.

In case the vaccine comprises a component of the virus, the “component”may be an immunogenic structure of the virus such as a subunit thereofincluding a chimeric subunit, or a nucleic acid fragment such as part ofthe genome of the virus. The component may also be recombinantly orsynthetically produced or modified.

Thus the vaccine may include whole viruses, the infectivity of which hasbeen inactivated, or subunit vaccines containing certain antigenicstructures, proteins or peptides of the virus, or their combination(such as virus like particles), or fragments of viral RNA or cDNAencoding the whole virus or individual viral proteins or inactivatedforms of the virus.

Inactivated vaccines may be produced by propagating the virus in cellcultures and by purifying it from infected cells and culture media byhigh-speed centrifugation in a density gradient formed by sucrose orother high-density media. Alternatively the virus may be purified bychromatography. The infectivity of the purified viruses is destroyed byinactivating the viruses by chemical treatment (e.g. formalininactivation like that used to produce inactivated polio virus vaccine),irradiation or heat treatment. Replication defective viruses may beprepared by physical or genetic inactivation e.g by deleting astructural gene in the viral genome, and producing a complementing cellline constitutively expressing the protein encoded by the gene deletedin order to replicate the defective virus.

Subunit vaccines may consist of purified viral proteins or recombinantviral proteins, synthetic peptides corresponding to viral antigenicepitopes, virus like particles or empty viral capsids, which areproduced during infection but lack the viral genome. These subunitvaccines can be administered either as such or conjugated to haptens orcarriers (e.g. ISCOM particles, chitosan, TLR agonists, biodegradablemicroparticles).

In case the vaccine comprises an antibody against at least one of saidenteroviruses, the antibody may be raised against the whole virus oragainst an immunogenic component thereof, or it may be recombinantlyproduced. The term antibody as used herein includes whole antibodymolecule of any isotype or fragments of antibodies such as Fab-fragmentsand scFv fragments.

The above mentioned vaccines can be given parenterally by injections(e.g. intramuscularly or subcutaneously), perorally, intradermally,transcutaneously, sublingually, intranasally, as inhalation, or perrectum. Each immunizing dose includes viral structures in a titer, whichis able to induce proper immune response in humans. This dose wouldcorrespond to that used in Salk-type inactivated poliovirus vaccineincluding 1.8-2 μg of viral protein per each dose and 20-40 antigenicD-units of poliovirus type 1,4-8 antigenic D-units of poliovirus type 2and 16-32 antigenic D-units of poliovirus type 3. The dose may also beanother, if it has been confirmed to be safe and immunogenic or able tostimulate the immune system.

The enteroviruses, their components or antibodies can be given indifferent combinations including one or more enterovirus serotypes givensimultaneously or serially. If the different enteroviruses, theircomponents or antibodies are given simultaneously the pharmaceuticalcomposition is in the form of a mixture of different enteroviruses,their components or antibodies.

The enteroviruses, their components or antibodies are used in themanufacture of a vaccine against T1D. They are formulated into apharmaceutical composition, which in addition to the active ingredientsthat elicit immune stimulation may comprise pharmaceutically acceptableexcipients, carriers, haptens and/or adjuvants. Excipients, carriers,haptens and adjuvants may include for example phenoxyethanol, magnesiumchloride, sucrose, thiomersal, formaldehyde, phenol, antibiotics(preservatives) or aluminium salts, polymer microparticles, ISCOMparticles, carrier proteins (e.g. cholera toxin), liposomes, proteinmicelles (haptens/adjuvants), or TLR agonists.

The pharmaceutical composition is preferably administered to a childwithin 5 years after birth, and more preferably within 3 years,especially within 2 years, and most preferably within 1 year afterbirth, with boosters given later in life to prevent or treat T1D. It canfor example be given at the age or 3, 6 or 12 months, with boosters atolder age. It can also be given to pregnant mothers to prevent T1D inthe baby, or prenatally to the pregnant mother and postnatally to thebaby. When given to pregnant mothers the induced antibody responseprotects the child because IgG class maternal antibodies are transferredto the fetus through the placenta and are thus protecting the childuntil the age of 6-12 months when maternal antibodies disappear from thechild's circulation. In addition, these protective antibodies aretransferred by breast-milk to the breastfed baby. These maternalantibodies, especially breast milk antibodies, have been shown toprotect against enterovirus infections in young infants (Sadeharju K,Knip M, Virtanen S M, Savilahti E, Tauriainen S, Koskela P, Akerblom HK, Hyoty H; Finnish TRIGR Study Group. Maternal antibodies in breastmilk protect the child from enterovirus infections. Pediatrics. 2007May; 119(5):941-6.) The vaccination regime can be used in the wholepopulation or in subpopulations carrying increased risk for T1D. Suchhigh-risk groups may include mothers or children with HLA-conferreddisease susceptibility to T1D, especially carriers of the HLA DR3 and/orDR4 alleles, subjects with type 1 diabetes in first or second-degreerelatives or children testing positive for two or morediabetes-associated autoantibodies.

The vaccines described may be used in preventing and treating type 1diabetes, in inducing an immune response against CBVs, and ineliminating the diabetogenic effect of CBVs in a subject in need thereofby vaccinating the subject with the pharmaceutical compositionsdescribed. The viruses are conveniently administered in inactivatedform, as subunits, as virus like particles (VLPs), or as nucleic acids.The prevention and treatment also encompasses use of the vaccine inpreventing the progression of prediabetes into diabetes, i.e. preventinginfection, or eradication of an ongoing infection inautoantibody-positive children.

If not otherwise indicated “type 1 diabetes” or “T1D” as used hereinrefers to the classic form of the disease, which is associated with theappearance of autoantibodies against pancreatic beta-cells. This diseasemay also be called “classic type 1 diabetes” to be distinguished from“fulminant type 1 diabetes”, which is a form of diabetes that isassociated with a macrophage dominated inflammatory process, which doesnot involve autoimmune antibodies.

Type 1 diabetes can be prevented or treated by antiviral treatment. Thistreatment may be, for instance, RNA interference based on a siRNAmethod, a pharmaceutical preventing the growth of the CBVs, an antibodyagainst the CBVs or its component, or a molecule preventing the virusfrom adhering to the cell, such as a soluble cell receptor, or it may bea vaccine against the CBVs. These treatments may prevent the developmentof a T1D disease or treat a disease that has already developed. A personin need of treatment or prevention is given an “anti-coxsackie B viruscomposition”, which is a composition containing an effective amount of apharmaceutically active anti-CBV substance and pharmaceuticallyacceptable carrier. The anti-CBV substance may be a virus medicament,such as a chemical drug, a cytokine such as interferone-alpha orinterferone-beta, siRNA or a peptide that prevents the interactionbetween the virus and the receptor, or a soluble receptor molecule, oranti-CBV antibodies.

The invention is illustrated by the following non-limiting examples.

Example 1

Seroneutralization Analyses

Neutralizing antibodies were analyzed against a wide panel of differententerovirus serotypes in the same serum sample which was the firstautoantibody positive sample taken during the prospective follow-up.Thus, this time point represents the initiation of the beta-celldamaging process. In addition, antibodies against those serotypes whichwere found to be interesting in this initial screening were measured atadditional time-points to study the timing of infections and theirrelationship with the initiation of the beta-cell damaging process.

Altogether, seroneutralization analyses have been performed using 42viruses and 522 serum/plasma samples (174 triplets, two control childrenfor each case child). The completeness of the analyses in this virus setvaries from 100% to 84% (Echo30) being for a majority of the viruses(35/40) more than 97.7%. In order to study the effect of the strainvariation, two serotypes (CBV4 and Echo3) have been analyzed using boththe freshly isolated wild type strains (wt) and corresponding reference(ATCC) strains (rs).

The viruses were analyzed in seroneutralization analyses usingautoantibody seroconversion date samples. The viruses were CAV4, CAV5,CAV10, CAV16, EV71, CAV9, CBV1, CBV2, CBV3, CBV4-wt, CBV4-rs, CBV5,CBV6, Echo1, Echo2, Echo3-wt, Echo3-rs, Echo4, Echo5, Echo6, Echo7,Echo9, Echo11, Echo12, Echo13, Echo14, Echo15, Echo17, Echo18, Echo19,Echo20, Echo21, Echo25, Echo26, Echo27, Echo29, Echo30, Echo32, Echo33,EV74, EV78 and EV94.

The seroneutralization analyses were mainly carried out using a plaqueneutralization assay. In this analysis the ability of the serum/plasmato inhibit a certain virus's ability to form plaques in cell layers hasbeen determined as compared to controls, in which fetal calf serum hasbeen used instead of human serum. In the analysis the inhibition hasbeen considered to be significant (positive result, ++) when it has beenmore than 85%. The inhibition range between 85-75% has been consideredas weaker positive (+) and inhibition of less than 75% has been judgedas a negative result. Because these analyses have been performed usingtwo different serum/plasma dilutions (¼ and 1/16) the results have beencombined using the classification shown below. A minority of theanalyses (such serotypes that did not form plaques) has been carried outusing microneutralization analysis, in which the ability of the virus tokill cells can be monitored by CPE and via crystal violet vitalstaining. Similar classification was used for the results ofmicroneutralization assay as for plaque inhibition assay.

Class 1:4 1:16 Max positive (0) ++ ++ Highly positive (1) ++ +Moderately positive (2) ++ − Positive (3) + − Negative (4) − −Seroneutralization Results

The raw data was exported to Stata package and analyzed usingconditional logistic regression models to evaluate the risk of certainserotypes to cause T1D. The results of the conditional logisticregression analysis are given as odds ratios (ORs). If the OR in certainanalysis is higher than 1 and the lower limit of the confidence interval(CI at the level of 95%) remains above 1 the virus can be considered asconferring risk for T1D. On the other hand, if both the OR and thehigher limit of the 95% CI are below 1, such a serotype appearsprotective against T1D. In such cases where OR is either over or below 1and also the 95% CI includes values on both side of 1, the result is notstatistically significant (P>0.05). In Table 1 the OR and CI values forCBV1, CBV3 and CBV6 are presented.

TABLE 1 The OR and CI values of CBV1, CBV3 and CBV6. The statisticallysignificant results are bolded. [OR (CI)] [OR (CI)] [OR (CI)] Virus 0-3vs. 4 0-1 vs. 2-4 0-2 vs. 4 0-1 vs. 4 CBV1 1.50 (1.02-2.23) 1.10 1.561.39 (0.65-1.87) (0.94-2.58) (0.68-2.85) CBV3 0.39 (0.18-0.82) 0.56 0.360.50 (0.24-1.34) (0.15-0.85) (021-1.20) CBV6 0.64 (0.41-0.97) 0.57 0.86049    (0.21-1.59) (050-1.51) (0.16-1.57)

The prevalence of antibodies (percentage of children having neutralizingantibodies against each virus serotype) was also calculated. FIG. 1shows the results of the CBV-subset. The data represent the wholecohort. Classes 0-3 vs. 4, ORs are shown in the upper panel, andseroprevalence in the lower panel. Statistically significant results aremarked with circles.

It was found that CBV1 is a clear risk serotype for T1D, while CBV3 andCBV6 appear protective. This association was first drawn fromseroneutralization results of a single cross-sectional time-pointrepresenting the first autoantibody positive sample, and were laterconfirmed in multi-time point analyses in which the timing of infectionswas analyzed in more detail. In these multi-time-point analyses theclearest result was the strong association of the CBV1 risk effect tothe six month time window immediately preceding the first detection oftype 1 diabetes related autoantibodies.

Multi-Time Point Seroneutralization Analysis with CBV1

To study the temporality of CBV1 infection with respect to AABappearance, the following time points were selected for theseroneutralization analyses:

-   -   12 months before autoantibody seroconversion    -   6 months before autoantibody seroconversion    -   point (first detection of autoantibodies; these samples were        analyzed previously)    -   12 months after autoantibody seroconversion    -   Time of the diagnosis of type 1 diabetes

According to this plan approximately 1 250 new samples fulfilling thecriteria above were identified, collected, anonymized and tested.

All samples were screened using ¼ and 1/16 serum dilutions. In thisreport the class comparison 03-vs. 4 and sensitivity analyses 1 and 2has been generally utilized, except in the tight criteria analyses(explained below) in which infections were diagnosed by subjectivejudgments done by two independent researchers on the basis of pre-fixedcriteria listed below for acute infection:

The acute infection was diagnosed according to following criteria, whichhad to be true for a classified infection:

-   -   A seroconversion from titer 0 to titer 4 or higher    -   The titer is 16 in at least one of the following samples    -   All follow-up samples remain positive

All analyses were done blindly without knowing the case-control statusof the child.

Antibodies to islet cells were detected by indirect immunofluorescence,and antibodies to insulin, glutamic acid decarboxylase, and proteintyrosine phosphatase-like protein (IA-2) were determined with specificradiobinding assays from a serum sample using standard methods. If twoof these four autoantibodies were detected, the subject was consideredautoantibody positive (Näntõ-Salonen et al., 2008. Lancet372:1746-1755).

Multi-Time Point Statistical Analyses

All statistical analyses presented are based on conditional logisticregression analyses. Three types of analyses were carried out for thisdata set. The timing of infections was determined and thetime-relationship between infections and appearance of autoantibodieswas analyzed. In other words, the frequency of infections among casechildren were compared to that in control children in each time windowseparately. The new time point results were analyzed similarly asdescribed above by comparing cases and controls in each time pointseparately. These analyses were carried out in the whole group and indifferent subgroups according to the following list:

-   -   Total data set from the whole cohort    -   Gender    -   Age    -   HLA genotype    -   Residence area    -   Combinations of different AABs in certain follow-up samples    -   Diagnosis of diabetes

Seroneutralization analyses of these three viruses have been carried outusing a plaque neutralization assay as described above.

Statistical analyses in loose and middle criteria approaches were doneaccording to combined classes 0-3 vs. 4 and also sensitivity analyses,in which class 3 (sensitivity analysis 1) or both classes 2 and 3(sensitivity analysis 2) are removed from the data set, were utilized.In the tight criteria approach the sensitivity analyses were not donedue to its intrinsic “sensitivity” character.

Timing of the Infections

The timing of infections caused by CBV1 and the other five relatedserotypes was approached in the following way: First, the recognizedinfections were categorized to different time windows relative to thedate of seroconversion to autoantibody positivity (AAB+ date). The usedwindows were as follows:

-   -   0. No infections or infection after the AAB+ date    -   1. Infection more than 12 months before the AAB+ date    -   2. Infection between 12 and 6 months before the AAB+ date    -   3. Infection within 6 months before the AAB+ date

In the loose criteria approach the first positive result was accepted asan infection regardless of the possibility of maternal antibodies or thepossibility that results of the later time point samples turn tonegative. The middle criteria approach was done similarly, except thatthose results that were biased by the identified maternal antibodieswere nullified. This judgement were done by two independent experts whoevaluated the data carefully and discarded those results in which thematernal antibodies could be the cause of the positivity. In the tightcriteria approach the similar judgement procedure were applied. In shortthe acute infection according to tight criteria were as follows:

-   -   a seroconversion from titer 0 to titer 4 or higher    -   the titer is 16 in at least one of the following samples    -   all follow-up samples remain positive

In addition to using these time-windows separately, analyses were alsoperformed by combining some of these windows together.

Data is shown for the two serotypes (CBV1 and CBV2) identified to beat-risk. The timing of infections caused by the CBV1 risk serotype inthese time windows is summarized in Tables 2 and 3. These analyses weredone using the loose criteria and middle criteria and the risk effect ofinfection was analyzed by comparing its frequency in differenttime-windows to that in window zero (see above). The basic statisticalanalyses i.e. classes 0-3 vs. 4. and sensitivity analyses 1 and 2 weredone.

TABLE 2 CBV1 timing conditional logistic regression analysis, otherwindows vs. window 0. Loose criteria approach with classes 0-3 vs. 4 andsensitivity analyses 1 and 2. Odds 95% CBV1 ratio P-value Conf. IntervalClass 0-3 vs. 4 Over 12 months before AAB+ date 1.77 0.064 0.97 3.2512-6 months before AAB+ date 2.24 0.007 1.25 4.00 6-0 months before AAB+date 3.56 <0.001 1.92 6.62 Sensitivity 1, Class 0-2 vs. 4 Over 12 monthsbefore AAB+ date 0.89 0.724 0.46 1.72 12-6 months before AAB+ date 1.250.468 0.68 2.31 6-0 months before AAB+ date 2.19 0.018 1.15 4.17Sensitivity 2, Class 0-1 vs. 4 Over 12 months before AAB+ date 1.190.684 0.51 2.79 12-6 months before AAB+ date 0.98 0.950 0.47 2.03 6-0months before AAB+ date 2.09 0.067 0.95 4.59

TABLE 3 CBV1 timing conditional logistic regression analysis, otherwindows vs. window 0. Middle criteria approach with classes 0-3 vs. 4and sensitivity analyses 1 and 2. Odds 95% CBV1 ratio P-value Conf.Interval Class 0-3 vs. 4 Over 12 months before AAB+ date 1.62 0.262 0.703.75 12-6 months before AAB+ date 0.77 0.417 0.41 1.44 6-0 months beforeAAB+ date 2.09 0.002 1.32 3.33 Sensitivity 1, Class 0-2 vs. 4 Over 12months before AAB+ date 1.14 0.795 0.41 3.17 12-6 months before AAB+date 0.66 0.307 0.30 1.46 6-0 months before AAB+ date 2.10 0.007 1.233.57 Sensitivity 2, Class 0-1 vs. 4 Over 12 months before AAB+ date 0.700.629 0.16 3.02 12-6 months before AAB+ date 0.36 0.078 0.11 1.12 6-0months before AAB+ date 1.44 0.342 0.68 3.05

Interestingly, in these analyses the CBV1 infections are clearly foundmore often in case children according to risk hypothesis with infectionsoccurring close to the time of autoantibody seroconversion (at the timewindow 6-0 months before the AAB+ date). The result is same in allanalyses, except that in sensitivity analysis 2 the statisticalsignificance is missed. Because in this analysis we have accepted allpositive antibody results (probably including false positive findings),except maternal antibodies in middle criteria analyses, it is essentialto compare these results to those obtained using tight criteria forinfections (Table 4).

TABLE 4 CBV1 timing conditional logistic regression analysis, otherwindows vs. window 0. Tight criteria approach. Odds 95% Conf. CBV1 ratioP-value Interval Over 12 months before AAB+ date 0.67 0.455 0.24 1.8912-6 months before AAB+ date 1.38 0.491 0.55 3.46  6-0 months beforeAAB+ date 3.76 0.001 1.68 8.44

In this analysis, a lot of data have been removed to exclude allpossible wrong positives. The results show that a credible dose responsecurve is observed highlighting the importance of the time window 6-0months before the AAB+ date and proving a definite risk effect of CBV1with convincing statistical outcome. Most importantly, because the same6-0 months before the AAB+ date window was observed to be statisticallysignificant in both the loose, middle and tight criteria approaches,these results cement this time period as a critical one for the CBV1infection as a risk to induce T1D. The tight criteria and loose criteriaresults are presented together in FIG. 2, which shows CBV1 ORs indifferent time windows before AAB+ date, Left: tight criteria, Right:loose criteria (class 0-3 vs. 4, sensitivity 1, sensitivity 2). The doseresponse curves can be seen with both approaches being the mostconspicuous in the tight criteria and loose criteria sensitivity 1analyses.

In one analysis we looked at the antibody responses of children aged 1.5years. In addition to the 1.5 year sample, also the cord blood sampleswere analyzed to study if the children had got protective antibodiespassively from their mothers. In this analysis it was found that if thechild was not protected by maternal antibodies and she/he got a CBV1infection within 1.5 years from birth, the risk effect of CBV1 wasextremely clear (OR=3.5, P<0.03) as shown in FIG. 3. This analysis wasdone using classification 0-3 vs. 4.

In order to understand the significance of this result let us interpretit in the terms of the population attributable risk (PAR) whichestimates the proportion of type 1 diabetes cases which could beprevented by the CBV1 vaccine in the population. Assuming that the OR is3.76 (=tight criteria result for window 6-0 months before the AAB+ date)and the prevalence of the CBV1 infection is 50% as indicated by theprevalence of CBV1 antibodies in AAB+ time point in control subjects,the PAR equation results in 58% for CBV1 alone (FIG. 4). FIG. 4 showsthe population attributable risk (PAR) for CBV1 calculated using an ORof 3.76 and a prevalence (Pc) of 50% representing the seroprevalence atthe time of autoantibody seroconversion in control subjects.

In the following analysis the time windows 6-0 and 12-6 were combined.The risk effect of CBV1 was significant also in this combined timewindow analysis (Table 5).

TABLE 5 CBV1 timing conditional logistic regression analysis, 12-0window against window 0. Tight criteria approach. Odds 95% Conf. CBV1ratio P-value Interval Over 12 months before AAB+ date 0.66 0.434 0.241.85 12-0 months before AAB+ date 2.46 0.004 1.33 4.53

Because the strongest risk effect was seen in the 6-0 window, this timeperiod was analyzed using another type of comparison. Instead ofcomparing it against window 0 (which was done in the previous analyses),this window was compared to all other windows in combination. Again, theprevious findings showing the critical importance of this time-windowwere supported by this analysis (Table 6).

TABLE 6 CBV1 timing conditional logistic regression analysis, 6-0 windowagainst combined other time windows. Tight criteria approach. Odds 95%Conf. CBV1 ratio P-value Interval 6-0 months before AAB+ date 3.78 0.0011.69 8.43Multi-Time Point Seroneutralization Analysis Further Including CBV2

In this analysis also CBV2 serotype was analyzed in similarmultitime-point setting as CBV1.

Timing of Infections

To clarify the possible timing of CBV subgroup infections, the same timepoints were selected for the seroneutralization analyses as describedabove.

The results presented below are based on the seroneutralization analysisof about 2 000 samples. In order to study the timing of infectionscaused by the six CBV serotypes the recognized infections werecategorized to different time windows relative to the date ofseroconversion to autoantibody positivity (AAB+ date). The used windowswere numbered from 0 to 3 as described above.

The same loose, middle and tight criteria approaches were used asdescribed above. Statistical analyses were done using conditionallogistic regression. Since some additional laboratory results have beenadded to the results are shown here also for CBV1. The results ofstatistical analyses for CBV serotypes are shown in Tables 7-8.

TABLE 7 CBV1 infection timing analysis. Loose criteria approach withclasses 0-3 vs. 4. Odds 95% Conf. CBV1 ratio P-value Interval Class 0-3vs. 4 Over 12 months before AAB+ date 1.89 0.035 1.05 3.40 12-6 monthsbefore AAB+ date 2.27 0.005 1.28 4.03  6-0 months before AAB+ date 3.54<0.001 1.95 6.42

TABLE 8 CBV2 infection timing analysis. Loose, Middle and Tight criteriaapproaches with classes 0-3 vs. 4. Odds 95% Conf. CBV2 ratio P-valueInterval Loose criteria Over 12 months before AAB+ date 0.79 0.441 0.441.43 12-6 months before AAB+ date 1.38 0.236 0.81 2.34  6-0 monthsbefore AAB+ date 2.32 0.001 1.40 3.84 Middle criteria Over 12 monthsbefore AAB+ date 0.80 0.523 0.41 1.58 12-6 months before AAB+ date 1.020.946 0.54 1.93  6-0 months before AAB+ date 2.29 0.001 1.43 3.66 TightCriteria Over 12 months before AAB+ date 0.88 0.838 0.26 3.00 12-6months before AAB+ date 1.00 0.994 0.33 3.03  6-0 months before AAB+date 2.44 0.059 0.97 6.15

Conclusions

In addition to CBV1 also the closely related CBV2 was found to be a riskvirus in multi-time-point analyses especially in the 6-0 months beforeAAB+ time window. Thus, the multi-time-point analyses produced a goodsecond candidate for the preventive vaccine.

PAR was also calculated for CBV2 using loose criteria OR=2.26 and theseroprevalence of 48% in background population, accordingly, we end upwith PAR=37% (FIG. 4). The result indicates that a vaccine cocktailcombination of CBV1 and CBV2 is most interesting. These two viruses canlead to a product having a major effect to prevent the onset of the type1 diabetes. Altogether, the results indicate that CBV2 is also a riskvirus associated with the pathogenesis of T1D and therefore useful inthe development of the vaccine.

Multi-Time Point Seroneutralization Analysis Further Including CBV6

In this analysis also CBV6 serotype was analyzed in a similarmultitime-point setting as CBV1 and CBV2. However, in this case, inaddition to the whole cohort analysis, the analyses were also doneseparately for each gender.

Timing of Infections

To clarify the possible timing of CBV subgroup infections, the same timepoints were selected for the seroneutralization analyses as describedabove.

The results presented below are based on the seroneutralization analysisof about 2 000 samples. In order to study the timing of infectionscaused by the CBV serotypes the recognized infections were categorizedto different time windows relative to the date of seroconversion toautoantibody positivity (AAB+ date). The used windows were numbered from0 to 3 as described above.

The same loose, middle and tight criteria approaches were used asdescribed above. Statistical analyses were done using conditionallogistic regression. The results of the statistical analyses for CBV6are shown in Table 9.

TABLE 9 CBV6 infection timing analysis for the whole cohort andseparately for females and males. Loose, Middle and Tight criteriaapproaches with classes 0-3 vs. 4. Loose Middle Tight CBV6 CBV6 CBV6Months before AAB+ OR P

OR P

OR P

Both gender >12 0.96 0.87 0.56 1.65 1.19 0.58 0.65 2.16 0.67 0.62 0.133.3 12-6  1.54 0.09 0.94 2.54 1.32 0.33 0.76 2.32 0.5 0.39 0.11 2.38 6-01.35 0.23 0.82 2.22 1.35 0.23 0.83 2.2 1.16 0.84 0.28 4.86 Girls >120.57 0.29 0.20 1.62 0.69 0.51 0.22 2.11 nd nd nd nd 12-6  2.14 0.06 0.964.77 1.39 0.47 0.57 3.39 nd nd nd nd 6-0 2.42 0.05 1.02 5.78 2.69 0.031.09 6.60 2.00 0.49 0.13 32.02 Boys >12 1.23 0.53 0.65 2.32 1.59 0.210.77 3.28 1.33 0.75 0.22 7.98 12-6  1.23 0.53 0.64 2.38 1.27 0.52 0.612.66 0.67 0.62 0.13 3.31 6-0 0.99 0.97 0.53 1.85 0.96 0.89 0.52 1.760.97 0.97 0.18 5.32

Conclusion

Although CBV6 was generally found as a protective serotype in AAB+single time point analysis, in multi-time point analyses it showed atrend for risk in the time window 12-6 months before AAB+ time window.Therefore the importance of CBV6 was studied in more detail and the sametiming analysis was carried out separately for females and males. Theresults of this analysis showed that the observed risk trend of the CBV6in the whole cohort associated strongly with female gender in both timewindows 12-6 months and 6-0 months before AAB+ time window. In contrast,for males there was no evidence of CBV6 associated risk. So, in overall,CBV6 may appear as a protective serotype, but when a certain subcohort,such as the females, is analyzed, it can turn into a risk virus in asaid subcohort.

Example 2

A CBV1 Inactivated Vaccine Protects Mice Against Viremia and Infectionof the Pancreas

Method

An inactivated vaccine was produced by amplification of CBV1 in Verocells, followed by sucrose purification and formol-inactivation. Thedose of the vaccine is expressed as the initial content in infectiousvirus (equivalent CCID₅₀).

BALB/c male and female mice, previously shown to be susceptible to CBV1infection, were immunized by the i.m. route with three injections offormol-inactived CBV1 at D0, D21 and D35. Two doses of the vaccine(4×10⁴ and 2×10⁵ eq CCID₅₀) were assessed for mice immunization. Oneweek after the last immunization, mice were challenged with live CBV1via the i.p. route. Viremia and infection of the pancreas were monitoredin immunized and control mice by CBV1 genomic titration of blood samplesand pancreas tissue homogenates.

Results

Immunogenicity of the vaccine was first evaluated by analyzing theinduction of neutralizing antibodies in individual mouse. The meanvalues obtained for each group of 5 mice are reported in Table 10.

Neutralizing antibodies were detectable in all immunized groups after 2injections (D35) whatever the vaccine dose used for immunization. Aftercompletion of the 3 vaccine administrations, all immunized mice showeddetectable neutralizing antibodies titers when using the 2×10⁵ eq CCID₅₀vaccine dose.

TABLE 10 An inactivated CBV1 vaccine induces consistent neutralizingantibodies in mice Vaccine dose Neutralizing antibody titers (eq CCID₅₀)D2 D21 D35 D42 ♂ 4 × 10⁴ < 1/20 < 1/20 1/20 1/44  2 × 10⁵ < 1/20 < 1/201/24 1/44* PBS (naive) < 1/20 < 1/20 < 1/20  < 1/20  ♀ 4 × 10⁴ < 1/20 <1/20 1/24 1/144 2 × 10⁵ < 1/20 < 1/20 1/28 1/76* PBS (naive) < 1/20 <1/20 < 1/20  < 1/20  *all mice responding

The protective effect of the vaccine was then assessed after a challengewith live CBV1 administered at an infectious dose previously shown toinduce a marked viremia at D2 and replication of the virus in pancreasup to D7.

In contrast to naive mice, all immunized mice were protected from CBV1infection as demonstrated by the absence of detectable virus in blood(FIG. 5) and in pancreas (FIG. 6) in the days following the ipchallenge.

Conclusion

The inactivated CBV1 vaccine protects against viremia and viralreplication in the pancreas caused by live CBV1. As induction ofautoimmunity following infection has been related to the inflammationcaused by viral replication (ref Horwitz et al, Nat Med 1998; ZiprisClin Immunol 2009), the CBV1 vaccine can protect from CBV1-inducedautoimmunity.

Example 3

Virus Infection and Diabetes in SOCS-1 Transgenic NOD Mice

A recently developed model for enterovirus-induced type 1 diabetes wasused with mice expressing the suppressor of cytokine signaling-1(SOCS-1) in pancreatic beta cells. By expressing SOCS-1 the pancreaticbeta cells lose their ability to respond to interferons and by that alsotheir ability to protect themselves against the virus. An analysis ofthe pancreata from infected SOCS-1 transgenic animals (here denotedSOCS-1-Tg NOD mice) demonstrate that diabetes is the result of beta celldestruction (Flodstrom, M. et al., 2002. Nat Immunol 3(4), 373-82;Flodstrom, M. et al., 2003. Diabetes 52(8), 2025-34).

Mice and Animal Husbandry:

All mice were breed and housed under specific pathogen free conditions.The SOCS-1-Tg NOD mice were bred as heterozygotes and screened for thepresence of the SOCS-1-transgene by PCR.

Virus Infections

Mice aged 8 to 10 weeks were infected with one intra-peritoneal (i.p.)injection of CBV1 (10¹, 10³-10⁸, and 4×10⁸ CCID50/animal). Weight andvenous blood glucose measurements were done on non-fasting mice with 1-2days intervals during the acute phase of the infection (app. day 3-12p.i) and thereafter on a weekly basis. Animals were considered diabeticwhen having two consecutive blood glucose measurements >13 mM. Diabeticanimals were sacrificed on the same day as the second reading was made.All nondiabetic animals were followed up to at least 21 days postinfection (p.i.). Pancreata were retrieved upon sacrifice and were fixedin 4% formalin for histology.

Histology and Immunohistochemistry:

Paraffin sections (4 μm) were made from selected pancreata and stainedwith hematoxylin and eosin (H&E) or with guinea pig anti-insulin or ratanti-glucagon primary antibodies (DAKO Cytomation, Stockholm, Sweden),as previously described in e.g. (Flodstrom et al., 2002 supra).

Summary of Obtained Results

According to plan, SOCS-1-Tg NOD mice were challenged with differentdoses of CBV1 (10¹, 10³-10⁸, and 4×10⁸ CCID50/animal). The infectionswere well tolerated with no or minor effects on animal weights. SomeSOCS-1-Tg NOD mice became diabetic rapidly after infection, with thehighest incidence (50%) obtained with the dose 4×10⁸ CCID50/animal(Table 11).

A histological analysis of selected pancreata demonstrated that micethat developed diabetes had lost most, but not all, of their beta cellmass. Some islet completely lacked insulin-positive cells while othersstill contained cells positively stained with the insulin-antibody.

Conclusions and Comments

CBV1 triggered diabetes in SOCS-1-Tg NOD mice although diseasepenetrance was at most 50% (4×10⁸ CCID50/animal, n=2). Diabetesappeared, at least in part, to be the result of a destruction of thepancreatic beta cells as animals that had developed diabetes had isletseither lacking insulin-positive beta cells or islets with lower thannormal numbers of insulin-positive cells. The islets in SOCS-1-Tg NODmice that did not develop diabetes appeared normal, some withsurrounding lymphocyte infiltrates that are commonly seen in SOCS-1-Tgand non-Tg NOD mice at this age.

TABLE 11 Incidence of diabetes in CBV1 infected SOCS-1-Tg NOD and NODmice SOCS-1-Tg NOD mice Diabetic Diabetic Virus dose animals animals No.of No. of (CCID50/-animal) of total (percent) females males 10¹ 0/6 0 60 10³ 0/5 0 3 2 10⁴ 1/6 17 2 4 10⁵ 1/7 14 7 0 10⁶ 1/8 13 8 0 10⁷ 2/7 290 7 10⁸ 2/5 40 0 5 4 × 10⁸ 1/2 50 0 2

Example 4

Risk Character of CBV2

In this study altogether 284 serum samples were collected from childrenat the age of one year (range 321-430 days) and their seroneutralizationresponses were studied. Seroneutralization results of autoantibodyseroconversion date done with six CBV serotypes were compared to resultsof the samples from type 1 diabetes diagnosis date taken from the samechildren and using the same viruses. In this analysis the referencegroup consisted of children who were negative for a given virus in bothsamples. The results are shown in Table 12. A link between CBV2infections and risk of type 1 diabetes was detected when the data ofinterval between autoantibody seroconversion date samples and thesamples taken at the date of diabetes diagnosis were compared. CBV2showed a risk trend with high OR indicating that it may accelerate theautoimmune process progression after its initiation.

TABLE 12 Conditional logistic regression analysis of 6 CBV serotypes.Sample interval between autoantibody seroconversion date and type 1diabetes diagnosis date were compared. Loose criteria approach: classes0-3 vs. 4. Changes from negative to positive have been analyzed againstreference group negative to negative. OR P 95% Conf. Interval CBV1 0.820.797 0.18 3.78 CBV2 6.04 0.106 0.68 53.32 CBV3 0.78 0.778 0.14 4.36CBV4 0.35 0.339 0.04 3.02 CBV5 0.18 0.108 0.023 1.45 CBV6 0.89 0.8130.35 2.27

Example 5

Antibody Cross-Reactivity within CBV Serotypes

Antibody cross-reactivity within the CBV serotypes was analyzed usingthe seroneutralization study cohort with two sample series. Theantibodies against CBV serotypes were measured in the plaqueseroneutralization assay and the serum samples were titrated to theirendpoint titer. Samples were analyzed from 23 children having antibodies(titer at least 1/16) against CBV1, CBV3 or CBV6. For the rest of theseviruses it was lower but at a detectable level (classes 1-3). The othersample series included eight children having one of the CBV serotypesdetected in a stool sample. In all eight children the antibody responsewas specifically induced with high titers (titer at least 1:512) againstthe serotype detected in stools. Here the serum sample was collectedimmediately before or straight after the CBV positive stool sample andthe antibody response remained high in the follow-up samples. When thechildren selected for these groups were observed as one group a cleartendency was observed. According to this cohort analyses CBV3 showedcross-reactivity with CBV1, CBV2 and CBV6 (Table 13).

TABLE 13 The main trends of the low-titer temporary peaks suggestingcross-reactivity between different CBV serotypes. CBV3 homotypicresponses were found in 18 children (from 65 samples) of whom 12children (in 26 samples) also had low-titer temporary responses in CBV1,CBV2 or CBV6. Number of Number of Low-titer samples where childrenhaving Homotypic temporary low-titer temporary low-titer temporaryresponse response in response response CBV3 CBV1 9 4 CBV2 4 4 CBV6 13 7

Another cross-neutralization assay was performed using monospecific seraraised against CBV1-6 serotypes in monkeys (specific strains produced bythe Swedish Institute for Communicable Diseases) and CBV1 and CBV3-6ATCC-reference strains in horses (purchased from ATCC). Analyses wereperformed with the CBV virus strains available. The cross-neutralizationanalyses performed with hyperimmunized monkey sera (antibody set no. 1)showed strong cross-reactivity between CBV4 virus and antisera raisedagainst all other CBV serotypes (Table 14). Further, the CBV1 antiserumcross-reacted weakly with CBV6 and, vice versa, the CBV6 antiserumshowed weak cross-reactivity with the CBV1 virus. Anothercross-neutralization study performed with the hyperimmunized horse sera(ATCC-sera) showed that the CBV6 antiserum cross-neutralized both CBV1and CBV3 viruses (Table 15). No reaction vice-versa was observed forCBV1 or CBV3 antisera against the CBV6 virus. No cross-reactivity wasseen with CBV4 either, which is in contrast to the results obtained withthe antibody set 1.

TABLE 14 The cross-neutralization study performed with thehyperimmunized monkey sera (antibody set no. 1 from the SwedishInstitute for Communicable Diseases, Stockholm, Sweden). 75% plaqueinhibition is used as a threshold value for inhibition. CBV4 CBV4 CBV1CBV2 CBV3 (WT) (ref. strain) CBV5 CBV6 V19 V20 V21 V22 V22r V23 V24 CBV11:8000* NO NO 1:2048 1:2048 1:8 1:16 Antiserum 4** 4 1000 500 CBV2 NO1:4000 NO 1:8 NO NO NO Antiserum 500 CBV3 NO NO 1:8000 1:2048 1:128 1:16NO Antiserum 4 62.5 500 CBV4 1:16 NO 1:4 1:8000 1:16000 1:4 NO Antiserum750*** 3000*** 3000*** CBV5 NO NO NO 1:2048 1:2048 1:8000 NO Antiserum 44 CBV6 1:4 NO NO 1:1024 1:256 NO 1:16000 Antiserum 4000 16 62.5 *Upperrow: Titer with serotype-specific antisera (homotypic response) **Lowerrow: How many times the serotype-specific titer is higher thannon-specific cross-reactive titer ***For calculating specific overnon-specific titer for CBV4 antisera the mean of specific titers fromtwo different CBV4 strains has been used NO = no detectedcross-reactivity

TABLE 15 The cross-neutralization study performed with hyperimmunizedhorse sera (ATCC origin). 75% plaque inhibition is used as a thresholdvalue for inhibition. CBV4 CBV4 CBV1 CBV2 CBV3 (WT) (ref. strain) CBV5CBV6 V19 V20 V21 V22 V22r V23 V24 CBV1 1:128* NO NO NO NO NO NOantiserum CBV3 NO NO 1:128 NO NO NO NO antiserum CBV4 NO NO NO 1:20481:2048 NO NO antiserum CBV5 NO NO NO NO NO 1:2048 1:32 antiserum 64**CBV6 1:128 NO 1:128 NO NO NO 1:8192 antiserum 64** 64** NO = no detectedcross-reactivity *Upper row: Titer with serotype-specific antisera(homotypic response) **Lower row: How many times the serotype-specifictiter is higher than non-specific cross-reactive titer

Example 6

Sequence Analyses

The aim of this study was to identify enterovirus serotypes causing type1 diabetes by direct sequence analysis from serum samples collected fromchildren who had been followed from birth until they turned positive fordiabetes-associated autoantibodies or developed clinical type 1diabetes. The study was based on serum and plasma samples collected in aprospective birth cohort study, which included two subcohorts of thechildren. Cohort 1 comprised children who have been screened forenterovirus RNA in previous academic studies carried out at theUniversity of Tampere. The aim was to sequence all enterovirus positivesamples detected in this cohort. Cohort 2 included the same children whowere screened for neutralizing enterovirus antibodies in the previousstudy. The aim was to screen these cohort 2 children for the presence ofenterovirus RNA in serum and to sequence all virus positive samples.

The sequencing was done with semi-nested RT-PCR using EV group VP1region specific primers as disclosed in Nix et al., J Clin Microbiol.2006 August; 44(8): 2698-704, and using EV group VP2 region specificprimers as disclosed in Nasri et al. J Clin Microbiol. 2007 August;45(8): 2370-9.

In these analyses the data from cohort 1 and cohort 2 were combined.This combined cohort included 1629 samples from 241 case children and4066 samples from 687 control children. Altogether 115 samples werepositive for enterovirus including 34 positive samples from the casechildren and 81 positive samples from the control children (Table 16).

TABLE 16 Number of analyzed (N) and enterovirus positive samples (EV+)of case and control groups in the combined cohort. Combined cohort 1Whole follow- Birth- 6 mo window prior AAb- and 2 up AAb Aab T1D Case(N) 4066 2668 1095 914 Control (N) 1629 1006 346 517 Case (EV+) 81 58 1816 Control EV (+) 34 20 9 9

Sequencing analyses targeted to altogether 91 enterovirus positivesamples in cohort 1 and 24 enterovirus positive samples in cohort 2. Outof these 115 positive samples, 34 were from case children and 81 fromcontrol children. These samples included 25 samples from case childrenin cohort 1 and 9 samples from case children in cohort 2, as well as 66enterovirus positive samples from control children in cohort 1 and 15enterovirus positive samples from control children in cohort 2. Sequenceanalyses were also done for some samples, which were excluded fromstatistical analysis of screening PCR results. All enterovirus sampleswere sequenced from VP1 and VP2 regions which allow the identificationof the serotype. Altogether 9 case samples (26%) and 26 control samples(28%) were sequenced successfully. All samples which were successful inboth VP1 and VP2 sequencing showed the same serotype in a blast search.In addition, all enterovirus positive samples were sequenced for the5-NCR sequence amplified by the screening RT-PCR.

The serotype distribution differed clearly between case and controlchildren: In the case children coxsackie B serotypes predominated andcoxsackie A serotypes were rare, while in the control children coxsackieA serotypes predominated. Among the case children the CBV virusesrepresented 44% of all serotypes identified, and among the controlchildren they represented only 19% of all identified serotypes (Table17). The CBV serotypes identified were CBV3, CBV4 and CBV5 in bothgroups.

TABLE 17 Proportion (%) of detected enterovirus types divided into twogroups: CBV viruses and non-CBV viruses. These results are based on VP1and VP2 sequencing in the case and control subjects. Virus group CasesControls CBV (%) 44 19 non-CBV (%) 56 81

Conclusion

The predominance of the CBV serotypes in the sera of case childrenindicates that the CBV group viruses are linked to T1D and play a rolein the pathogenesis of the disease. This result together with thestrongest endpoint (children progressed to clinical type 1 diabetes incohort 1), i.e. statistically significant risk of enterovirus infectionsin the 6 month window before autoantibody seroconversion date, alsoindicate a causal linkage between the CBV infections and the initiationof the type 1 autoimmune process.

The invention claimed is:
 1. A method of inducing an immune response tocoxsackie B virus (CBV) in an individual, the method comprisingadministering to the individual an effective amount of a compositioncomprising: a) CBV1 and CBV2 in inactivated form or in the form of asubunit or a virus-like particle (VLP); and b) at least one CBV selectedfrom CBV3, CBV4, and CBV5, in inactivated form or in the form of asubunit or a VLP, wherein the composition does not include CBV6, whereinsaid administering induces an immune response to CBV in the individual.2. The method of claim 1, wherein the CBVs are inactivated.
 3. Themethod of claim 1, wherein the CBVs are in the form of a subunit of theCBVs.
 4. The method of claim 1, wherein the CBVs are in the form of VLPsof the CBVs.
 5. The method of claim 1, wherein the composition comprisesCBV1, CBV2, CBV3, CBV4, and CBV5 in inactivated form or in the form of asubunit or a VLP.
 6. The method of claim 1, wherein the compositioncomprises a pharmaceutically acceptable excipient.
 7. The method ofclaim 1, wherein the composition comprises an adjuvant.
 8. The method ofclaim 1, wherein the composition is administered intramuscularly.
 9. Themethod of claim 1, wherein the composition is administeredsubcutaneously.